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Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus
International Immunopharmacology 35 (2016) 265–271
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Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus Zhilin Qi a,b,⁎, Shimei Qi a,b, Liefeng Ling a,b, Jun Lv a,b, Zunyong Feng b a b
Department of Biochemistry, Wannan Medical College, Wuhu, Anhui, China Anhui Province Key Laboratory of Active Biological Macro-molecules, Wuhu, Anhui, China
a r t i c l e
i n f o
Article history: Received 5 January 2016 Received in revised form 16 March 2016 Accepted 1 April 2016 Available online xxxx Keywords: Salidroside LPS JAK2-STAT3 Inflammation Acute lung injury
a b s t r a c t Salidroside (SAL) is an active ingredient isolated from the Rhodiola rosea, has potent anti-inflammatory effect, but the mechanism is still elusive. The purpose of this study is to verify the effects of SAL on LPS-induced inflammatory response and investigate the possible underlying molecular mechanism. RAW264.7 cells were preincubated with SAL for 2 h, then stimulated with or without LPS for another 16 h. The levels of TNF-α, MCP-1, IL-6, and PGE2 were detected by ELISA, and the production of NO was determined by nitrite analysis. The expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blotting. In RAW264.7 cells and murine peritoneal macrophages, the activation of signal molecules was also measured by Western blot. The nuclear translocation of STAT3 was determined by Laser confocal and nucleocytoplasmic separation experiments. Our results showed that SAL attenuated the productions of TNF-α, IL-6, MCP-1, PGE2 and NO dose dependently. SAL also suppressed LPS-induced expressions of iNOS and COX-2 significantly. Further studies revealed that SAL down-regulated the phosphorylation of JAK2-STAT3 signaling pathway and reduced the nuclear translocation of STAT3 induced by LPS in RAW264.7 cells and primary peritoneal macrophages. In addition, consistent with the results in vitro, in the model of mice acute lung injury (ALI) induced by LPS, SAL reduced the infiltration of inflammatory cells and decreased the levels of serum TNF-α and IL-6 obviously. Taken together, these data indicated that SAL exerted anti-inflammatory action via down-regulating LPS-induced activation of JAK2-STAT3 pathway and suppressing STAT3 transfer into the nucleus at least in part. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Lipopolysaccharide (LPS) also known as endotoxin, which is the main component of Gram-negative bacteria cell wall. Once released in the circulation, LPS can induce various host cells infection, especially mononuclear phagocytes that can produce pro-inflammatory cytokines including TNF-α, IL-6, and other inflammatory mediators, such as NO and PGE2, which is the production of iNOS and COX-2, respectively [1–3]. It has been reported that many inflammation related diseases are closely relevant with the accumulation of pro-inflammatory cytokines and mediators [4]. So, suppression the release of inflammatory cytokines and mediators may be an important strategy for treatment many inflammatory disorders. The JAK-STATs (Janus kinase-signal transducers and activators of transcription) is an important signaling pathway activated by many extracellular signaling ligands including LPS [2,5]. The signaling pathway has variety of biological effects such as cell proliferation, apoptosis, differentiation, and inflammation [6]. The binding of ligands and their ⁎ Corresponding author at: Department of Biochemistry, Wannan Medical College, No. 22 Wenchang West Road, Wuhu, Anhui 241002, China. E-mail address: [email protected] (Z. Qi).
http://dx.doi.org/10.1016/j.intimp.2016.04.004 1567-5769/© 2016 Elsevier B.V. All rights reserved.
receptors can induce the phosphorylations of JAKs and its downstream transcription factors STATs. Phosphorylated STATs form homo or heterodimers, transfer into the nucleus to regulate the expression of target genes associated with inflammation such as iNOS and COX-2 [7]. As we known, inflammation plays a key role in acute lung injury (ALI), and JAK-STATs signaling pathway plays an important role in the inflammation process of acute lung injury (ALI) [8]. Due to the critical role of JAKs-STATs signaling pathway in inflammatory response, inhibition the signaling pathway activation may be an interested strategy for the treatment of inflammation related diseases. Salidroside, (SAL, p-hydroxyphenethyl-β-D-glucoside), a major bioactive component extracted from Rhodiola rosea [9]. Recently, increasing studies have demonstrated that salidroside had a variety of pharmacological functions including anti-inflammatory [1,3], antitumor [10,11], neuroprotection [12,13]. The anti-inflammatory effect of salidroside on attenuation the expressions of iNOS and COX-2 and the release of inflammatory cytokines have been reported, the potent molecular mechanisms involving the inhibition of MAPKs and NF-κB signaling pathways activation [1,14]. Although salidroside has been shown to reduce inflammatory response in RAW264.7 cells upon LPS stimuli, the more detailed molecular mechanisms underlying the antiinflammatory action have not been evaluated yet. In addition, STATs
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have been implicated to be key transcription factors in both immunity and inflammatory pathways [15,16]. Thus, the aim of this study is to explore whether the anti-inflammatory activity of salidroside is mediated via suppressing JAK-STATs signal pathway activation upon LPS stimulation in RAW264.7 cells and peritoneal macrophages. In the present study, we first verified the anti-inflammatory activity of salidroside and then investigated the influence of salidroside on the activation of JAK-STATs signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Secondly, we detected the protective effects of salidroside on LPS-induced mice acute lung injury (ALI). Our results showed that salidroside could attenuate LPSinduced release of pro-inflammatory cytokines and mediators, alleviate LPS-induced ALI through suppressing of JAK2-STAT3 signal pathway and preventing the nuclear translocation of STAT3. Taken together, our study provided new sight in the anti-inflammatory mechanism of salidroside.
AG490 (20 mg/kg) 2 h prior to LPS (25 mg/kg, i.p.) treatment 12 h. After ALI, blood was collected to determine the serum levels of IL-6 and TNF-α from rats sacrificed under sodium pentobarbital (30 mg/kg, i.p.) anesthesia. In parallel experiments, after LPS stimulation for 12 h, the lungs were fixed with paraformaldehyde and then excised from rats sacrificed under sodium pentobarbital anesthesia.
2. Materials and methods
RAW264.7 cells were planted in 12-well plates and then preinclutured with different concentrations of salidroside for 2 h before 100 ng/mL LPS stimulation for another 16 h. The levels of IL-6, TNF-α, MCP-1, PGE2 in the cell culture supernatants, and the amounts of IL-6, TNF-α in the serum of rats were detected by using ELISA kit, following the manufacture's instructions. Each of the different treatments was repeated three times.
2.1. Reagents and antibodies Salidroside (purity, N 95%) was purchased from Aladdin Industrial Corporation (Shanghai, China). LPS was obtained from Sigma. 4,6diamidino-2-phenylindole (DAPI) was obtained from Invitrogen (Carlsbad, CA). The antibodies against phospho-JAK1(Y1022/1023), phospho-JAK2(Y1007/1008), JAK1, JAK2, phospho-STAT1 (Tyr701), STAT1, STAT3, COX-2, iNOS and GAPDH were all from Cell Signaling Technology (Beverly, MA, USA). The antibody against phosphoSTAT3 (Tyr705) was gained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies used for Western blotting were purchased from Rockland Immunochemical. Alexa Fluro 555 goat anti-rabbit IgG used in Laser confocal experiment was obtained from Invitrogen. 2.2. Cell culture RAW264.7 cells, purchased from Cell Bank of the Kunming (Kunming, China). were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) added with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin. 2.3. Murine peritoneal microphages extraction and culture Murine peritoneal macrophages were extracted from BALB/c mice based on the previous described method [17]. BALB/c mice (2-month-old females weighing 20 ± 2 g) were purchased from Nanjing University Experimental Animal Center and anesthetized with 2% isoflurane in oxygen via a facemask. DMEM (5–7 mL) were intraperitoneally injected, the abdomen of mice was kneaded 2– 3 min gently, then the peritoneal lavage fluid was extracted and centrifuged at 1000 rpm for 5 min at room temperature. After 2 times washing with PBS, the cells were resuspended in DMEM with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. All the cells were cultivated in six-well plates and cultured at 37 °C in 5% CO 2 . After 4 h incubation, we changed the media, removed the non-adherent cells, the cells in six-plates were used for experiment. 2.4. Animal model of acute lung injury (ALI) All procedures were approved by the Animal Care and Use Committee of Wannan Medical College. Animals were handled in accordance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. In this experiment, ALI was induced by LPS in Wistar rats (180–220 g). In briefly, rats were administered intraperitoneally with salidroside (20 mg/kg) or
2.5. Histological examination Lung tissues were rinsed thoroughly in PBS, fixed in 4% formalin, embedded in paraffin. Then the sections were stained with hematoxylin and eosin (H&E) for histological examination. Three parameters including congestion, edema and infiltration of inflammatory cells were used to evaluate the Lung injuries induced by LPS [18]. 2.6. Determination of inflammatory cytokines and medicators
2.7. Cell viability assays RAW264.7 cells were planted into 96-well plates at a density of 5000–6000 per well 24 h before treatment. Cells were treated with different dose of salidroside for 24 h, cell viability was determined by using the Cell Counting Kit-8 (Kaiji, Nanjing, China). Briefly, cells were treated with salidroside for the indicated time and then followed by incubating with 10 μL CCK-8 working solution at 37 °C and 5% CO2 for 2 h. The absorbance of each well at 450 nm was measured using MULTISKAN GO (Thermo). Each of the different treatments was done for three times, the data were presented with mean ± SD. 2.8. Nitrite analysis Cells were seeded in 96-well plates and then pre-treated with salidroside for 2 h prior to LPS stimulation or not. NO synthesis was determined spectrophotometrically by assaying the culture supernatants using the Griess reagent (Nanjing Jiancheng Bioengineering Institute). Absorbance was measured at 550 nm and the experiments were done in triplicate. 2.9. Western blot Briefly, RAW264.7 cells were rinsed with ice-cold PBS, and lysed for 30 min on ice in lysis buffer (Beyotime Biotechnology, China) supplemented with complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Lysates were centrifuged (1,2500 rpm) at 4 °C for 15 min. The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Equal amounts of protein were denatured and electrophoresed on 12% SDS-PAGE and followed by transferring onto nitrocellulose membranes (Whatman, GE Healthcare, NJ, USA). The proteins were visualized by the LI-COR Odyssey Infrared Imaging System (LI-COR) using IRDye800 flurophore conjugated secondary antibody. 2.10. Nuclear and cytoplasmic extraction RAW264.7 cells were treated with salidroside for 2 h and then stimulated with LPS or not for another 4 h. To separate the cytoplasmic and nuclear proteins, cell pellets were processed using the NE-PER nuclear
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and cytoplasmic extraction kit (Promega) according to the manufacturer's instructions.
2.11. Confocal microscopy RAW264.7 cells and primary macrophages were seeded in laser confocal small dish at 30% confluence, cells were pretreated with salidroside (200 μg/mL)for 2 h followed by LPS stimulation for another 4 h. Cells were washed with PBS, fixed with 4% polyformaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 3% bovine serum albumin in PBS for 1 h, the cells were incubated with STAT3 primary antibody overnight at 4 °C. After rinsing with PBS for three times, the cells were incubated with secondary fluorescent antibody (goat anti-rabbit IgG Alexa fluor 555 conjugates) for 1 h in the dark. After three times washing with PBS, the cells were nuclear-stained with DAPI for 3 min. Images were captured using LEICA TCS SP8 microscope.
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2.12. Statistical analysis Statistical analysis was performed with one way (ANOVA). Data were showed as means ± SD. Statistical calculations were performed by SPSS17.0. P b 0.05 was considered significant. 3. Results 3.1. Salidroside reduced the levels of pro-inflammatory cytokines and medicators induced by LPS in RAW264.7 cells As we know, the inflammatory cytokines and mediators such as TNF-α, IL-6, MCP-1, and PGE2 play an important role in inflammation. To verify the anti-inflammatory effect of salidroside on LPS induced inflammatory response, we first detected the release of IL-6, TNF-α, NO and PGE2 in LPS induced RAW264.7 cells. Fig 1A–E showed that LPS treatment obviously enhanced the levels of TNF-α, IL-6, MCP-1, PGE2
Fig. 1. Salidroside reduced the release of pro-inflammatory cytokines and mediators induced by LPS in RAW264.7 cells. (A–E) Cells were pre-incubated with salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with LPS (100 ng/mL) for another 16 h. The levels of TNF-α, IL-6, MCP-1, NO, and PGE2 were measured in the culture medium by ELISA kits or Griess reagents, respectively. (F) RAW264.7 cells were stimulated with different concentrations of salidroside for 24 h. Cell viability was evaluated using CCK-8 kit. The data were expressed as mean ± SD of three independent experiments. *P b 0.05 and **P b 0.01, versus control group.
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and NO. But, the cells pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL), the release of these cytokines and medicators was inhibited significantly in a dose dependent manner. In order to exclude the cytotoxicity of salidroside on cells, we detected the cytotoxic activity of salidroside by the CCK-8 assay. Fig 1F showed that salidroside had no cytotoxicity on cell viability even at the high dose of 400 μg/mL. 3.2. Salidroside reduced LPS-induced expressions of iNOS and COX-2 in RAW264.7 cells NO and PGE2 are two of important inflammatory mediators, which is the product of iNOS and COX-2 respectively. As above shown, salidroside attenuated the release of NO and PGE2 induced by LPS obviously. Thus, we further determined the role of salidroside on the levels of iNOS and COX-2. RAW264.7 cells were stimulated with salidroside for 2 h, then treated with LPS or not for 16 h, after the indicated time, the total proteins were extracted and subjected to Western blotting. As shown in Fig 2, LPS could induce a dramatic increase of iNOS and COX-2 proteins, while pre-treatment with salidroside significantly reduced LPS-elevated expressions of iNOS and COX-2 dose dependently. 3.3. Salidroside suppressed LPS-induced JAK2-STAT3 signal activation in RAW264.7 cells It has been reported that the STATs signaling pathway played important roles in inflammatory signaling cascades induced by LPS and other cytokines [19]. After activation, STATs form dimers, transfer into the nucleus and activate transcription of several target genes including iNOS [20]. To investigate whether the inhibitory effects of salidroside on the expressions of pro-inflammation cytokines and mediators are mediated via STATs pathway, we determined the effects of salidroside on the activation of STATs signal induced by LPS. Our previous study showed that, RAW264.7 cells stimulated with LPS, the STAT1 and STAT3 could be phosphorylated at 30 min and peaked at 4 h [2,7]. Thus, we detected the STATs phosphorylation at 4 h after LPS stimulation. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with 100 ng/mL LPS for another 4 h, Western blotting detected the phosphorylations of STAT1 and STAT3. As shown in Fig 3A, the phosphorylations of STAT1 and STAT3 were increased obviously after LPS stimulation. By contrast, the enhanced phosphorylation of STAT3 but not STAT1 was attenuated by salidroside in a dose-dependent manner. It has been reported that the STATs transcription factors were activated by their upstream kinases Janus kinases (JAKs) [6], we thus examined the effects of salidroside on the LPS-induced activation of JAKs. Our previous study showed that LPS stimulated RAW264.7 cells for 30 min, the phosphorylations of JAKs reached the maximum [2]. Then we determined the activation of JAKs after LPS stimulation for 30 min combined with or without salidroside. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, and then stimulated
Fig. 2. Salidroside suppressed the expressions of iNOS and COX-2 induced by LPS in RAW264.7 cells. Cells were pre-treated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with LPS (100 ng/mL) for 16 h. Cell lysates were collected, the levels of iNOS and COX2 were determined by Western blotting.
with LPS for 30 min, the phosphorylations of JAKs were analyzed by Western blotting. The pre-treatment with salidroside apparently attenuated LPS-induced JAK2 phosphorylation dose dependently, which was in accordance with the reduction of STAT3 phosphorylation. However, the phosphorylation of JAK1 induced by LPS was not affected by salidroside (Fig 3B). 3.4. Salidroside reduced LPS-induced nuclear translocation of STAT3 in RAW264.7 cells Since activated STAT3 should dimerize and translocate into the nucleus to regulate the transcription of target genes, we further explored whether salidroside could suppress the nuclear translocation of STAT3. Confocal microscopy experiment was used to detect the distribution of STAT3, nucleocytoplasmic separation experiment was used to confirm the level of STAT3 in the cytoplasm and nuclei. Fig 3C-D showed that STAT3 transferred into nucleus upon LPS stimulation. The cells pre-treated with salidroside, the accumulation of STAT3 in nuclei was inhibited evidently. Taken together, our results indicated that salidroside reduced LPS-induced inflammatory responses through inhibiting the activation and nuclear translocation of STAT3 in RAW264.7 cells. 3.5. Salidroside inhibited LPS-induced JAK2-STAT3 signal activation and suppressed the nuclear translocation of STAT3 in primary macrophages To further prove the role of JAK2-STAT3 signal pathway on the antiinflammatory of salidroside, we detected the effects of salidroside on LPS-induced JAK2-STAT3 activation and STAT3 nuclear translocation in murine peritoneal macrophages. Primary macrophages were preincubated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with 100 ng/mL LPS for 4 h or 30 min, The phosphorylations of JAK2 and STAT3 were detected by Western blotting. Consistent with the results of Fig 3A–B, salidroside could suppress LPS-induced JAK2-STAT3 signal pathway activation in a dose dependent manner (Fig 4A–B). In addition, we also detected the phosphorylations of JAK1 and STAT1 in primary macrophages. Our data showed that salidroside had no inhibitory effects on the phosphorylations of JAK1 and STAT1 induced by LPS (Fig 4A–B), which also consistent with the results of Fig 3A–B. Then, we detected the nuclear translocation of STAT3 in murine peritoneal macrophages. Cells were pre-treated with 200 μg/mL salidroside for 2 h, then stimulated with LPS for 4 h, the distribution of STAT3 was observed by confocal microscopy. Fig 4C showed that pre-treatment with salidroside could significantly reduce the nuclear translocation of STAT3 compared with the LPS group. 3.6. Salidroside inhibited LPS-induced acute lung injury To further certified the protective effects of salidroside on LPS induced inflammatory response, we detected the productions of serum IL-6 and TNF-α, observed the histological damage and inflammatory response in LPS induced acute lung injury model. Consistent with the results in vitro, the rats pre-treated with salidroside (20 mg/kg), the levels of serum TNF-α and IL-6 were obviously suppressed compared with the rats stimulated with LPS (Fig 5A–B). Histopathological examination showed that the histological damage, edema and inflammatory cell infiltration were alleviated obviously in the rats pre-treated with salidroside compared with the rats treated with LPS (Fig 5C). To address the relation of ALI and JAK2-STAT3 signal pathway, we used the AG490 (Sigma), JAK2 specific inhibitor as positive control. Fig 5C showed that pre-treated with AG490 (20 mg/kg), the histological damage, edema and inflammatory cell infiltration were alleviated obviously compared with the rats stimulated with LPS. Taken together, our results showed that the molecular mechanism of salidroside on LPS induced inflammatory response via suppressing JAK2-STAT3 signaling pathway in vivo and in vitro.
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Fig. 3. Salidroside inhibited LPS induced JAK2-STAT3 signal pathway activation and STAT3 nuclear translocation in RAW264.7 cells. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with LPS (100 ng/mL) for the 4 h (A, C, D) or 30 min (B). The phosphorylations of STAT1, STAT3, JAK1, JAK2 and total STAT1, STAT3, JAK1, JAK2 were determined by Western blotting (A–B). (C) Nuclear and cytoplasm fractions were extracted and detected the level of STAT3 by Western blot. (D) The nuclear translocation of STAT3 was determined by confocal microscopy. Cells were fixed with 4% paraformaldehyde, permeabilized with Trixon-100, incubated with antiSTAT3 antibody, and were assessed by immunofluorescence staining with Alexa Fluro 555 goat anti-rabbit IgG antibody (red). Cell nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion As an important bioactive ingredient, Salidroside has been widely used in Chinese traditional medicine [14]. It has been reported that salidroside had a lot of pharmacological effects including anti-
inflammatory. Although recent reports have shown that salidroside played its anti-inflammatory role through inhibiting the activation of MAPKs and NF-κB signal pathways [14], more detailed mechanisms were still unclear. It has been reported that JAK-STATs signaling pathway contributed to LPS-mediated inflammatory response in lung [3].
Fig. 4. Salidroside inhibited LPS induced JAK2-STAT3 signal pathway activation and STAT3 nuclear translocation in primary macrophages. Murine peritoneal macrophages were preincubated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with 100 ng/mL LPS for 4 h (A, C) or 30 min (B), The phosphorylations of STAT1, STAT3, JAK1 and JAK2 were detected by Western blot (A–B). (C) The nuclear translocation of STAT3 was determined by confocal microscopy. Cells were fixed with paraformaldehyde, permeabilized with Trixon100, incubated with anti-STAT3 antibody, and were assessed by immunofluorescence staining with Alexa Fluro 555 goat anti-rabbit IgG (red) antibody. Cell nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Salidroside inhibited the productions of serum pro-inflammatory cytokines and attenuated the acute lung injury induced by LPS in rats. Rats were administered intraperitoneally with salidroside (20 mg/kg) or AG490 (20 mg/kg) 2 h prior to LPS (25 mg/kg, i.p.) challenge. (A–B) Blood was collected after ALI (12 h after LPS stimulation), the serum levels of IL-6 and TNF-α from rats sacrificed under sodium pentobarbital anesthesia were detected by ELISA kits. (n = 5 rats/group). Data were expressed as mean ± SD. *P b 0.05 and **P b 0.01, versus control group. (C) Lung tissues from different group rats were collected and subjected to H﹠E staining and detected by light microscopy (magnification, ×400).
So, in the present study, we investigated the possible correlation between the anti-inflammatory effect of salidroside and JAK-STATs signaling pathway. Our data showed that salidroside significantly inhibited the activation of JAK2-STAT3 signaling pathway, and suppressed the nuclear translocation of STAT3. In this study, we first verified the anti-inflammatory effect of salidroside in RAW264.7 cells, we observed that salidroside suppressed the release of TNF-α, IL-6, MCP-1, PGE2, NO and downregulated the expressions of iNOS and COX-2, which were induced by LPS. In addition, the inhibitory effect was concentration dependent (Figs. 1 and 2). Secondly, we investigated the underlying antiinflammatory molecular mechanisms of salidroside in RAW264.7 cells.
Accumulating evidences had showed that a variety of signal pathways involved in the protective effects of salidroside such as NF-κB, MAPKs and PI3K-AKT signaling [21–24]. Some studies have proved that STATs and its upstream kinases played an important role in inflammatory and immune response induced by LPS [25,26]. But, whether the signaling pathway involved in the anti-inflammatory effect of salidroside is still unclear. Our previous study showed that STAT1 and STAT3 could be phosphorylated by LPS in RAW264.7 cells [2]. After activation, STATs dimerized and translocated to the nucleus to regulate the transcription of target genes [20]. In RW264.7 cells, we found that the pre-treatment with salidroside attenuated LPS induced STAT3 phosphorylation in a dose dependent manner (Fig 3A). But, the phosphorylation of STAT1 was not affected by salidroside. Because STATs
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phosphorylation is required for their nuclear translocations and transcriptional activities [19], to demonstrate whether salidroside could suppress the nuclear translocation of STAT3, we therefore determined the distribution of STAT3 in nuclear and cytoplasm. Western blotting detected the levels of nuclear and cytoplasm proteins in RAW264.7 cells. As shown in Fig 3C, STAT3 transferred into nucleus upon LPS stimulation. However, pre-treatment with salidroside could reduce the nuclear translocation of STAT3 induced by LPS dose dependently. Confocal microscopy analysis also revealed that salidroside attenuated the STAT3 nuclear translocation clearly (Fig 3D). These results proved that salidroside could inhibit LPS-induced inflammatory response via suppressing the activation of STAT3 at least partly. We further detected the activation of JAKs, which were the upstream kinases of STAT3. Our results showed that phosphorylation of JAK2 but not JAK1 was obviously inhibited by salidroside dose dependently in LPS stimulated RAW264.7 cells (Fig 3B). Our data suggested that salidroside attenuated LPS-induced inflammatory response via suppressing the activation of JAK2-STAT3 signaling pathway. To further determine the inhibitory effect of salidroside on JAK2STAT3 signaling pathway, we detected the activation of JAK2-STAT3 signal in primary macrophages. Consistent with the results in RAW264.7 cells, Fig 4A–B showed that pretreatment with salidroside, the phosphorylations of JAK2 and STAT3 were reduced obviously. The nuclear translocation of STAT3 was also detected by confocal laser experiment in murine peritoneal macrophages. Fig 4C showed that salidroside could inhibit the nuclear translocation of STAT3. It has been reported that pre-treatment with salidroside then stimulation with LPS could attenuate inflammatory cells infiltration, interstitial edema, hemorrhage compared with LPS stimuli in lungs of mice. In addition, salidroside could significantly reduce TNF-α, IL-6, and IL-1β levels in BALF [17]. Consistent with their results, our data also showed that salidroside could reduce the inflammatory cell infiltration, attenuate the productions of serum inflammatory cytokines TNF-α and IL-6 (Fig 5A–B). Mechanistically, LPS stimulated inflammatory signaling via TLR4, resulting in potent activation of MAPKs and NF-κB. These pathways played important roles in LPS-mediated tissue damage. The results of Guan et al. have shown that salidroside inhibited the LPS-induced ALI via NF-κB signal pathway [17]. But, the study of Mariano Severgnini, et al. demonstrated that STATs and their upstream kinases, Src and JAKs were activated in lungs of mice after LPS stimulation [25]. Their research suggested that JAK-STATs signaling pathway played an important role in LPS induced ALI. In addition, it has been reported that AG490, a special inhibitor of JAK2 pretreatment ameliorated histopathologic changes and alleviated edema in trauma-induced ALI significantly [18]. These evidences suggested that LPS-induced ALI and JAK-STATs signaling pathway were mechanistically linked. To further determine the link between the JAK2-STAT3 signaling pathway and ALI, we used AG490 as positive control, consistent with their results, in Fig 5C showed that pre-treatment with AG490, the inflammatory cell infiltration and pulmonary edema were alleviated obviously compared with the LPS treatment. Similarly to AG490, salidroside was shown to significantly reduce the inflammatory response in lungs of ALI rats induced by LPS. Taken together, our results proved that salidroside had protective effects in LPS induced inflammatory response in vivo and in vitro. In summary, the present study certified that salidroside exhibited its anti-inflammatory effect via inhibiting JAK2-STAT3 signaling pathway activation and STAT3 nuclear translocation. This study provided a new perspective for the anti-inflammatory function of salidroside. Acknowledgements The work was supported by College Outstanding Young Talent Support Program Key Projects (No. gxyqZD2016173), Natural Science Research Project of Anhui Colleges and Universities (No.·KJ2016SD59), the Natural Science Research Project of Anhui Provincial Education Department (No.·KJ2013B311), National Natural Science Foundation of
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China (31301171), The Key Scientific Research Project of Wannan Medical Colleges (WK2015Z01), Wannan Medical College Doctoral Fund, Anhui Province Key Laboratory Of Active Biological Macro-Molecules (No. 1306C083008).
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Shen, et al., A dual-role of Gu-4 in suppressing HMGB1 secretion and blocking HMGB1 pro-inflammatory activity during inflammation, PLoS ONE 9 (2014), e89634. [19] P.J. Murray, The JAK-STAT signaling pathway: input and output integration, J. Immunol. 178 (2007) 2623–2629 (Baltimore, Md: 1950). [20] D.E. Levy, J.E. Darnell Jr., Stats: transcriptional control and biological impact, Nat. Rev. Mol. Cell Biol. 3 (2002) 651–662. [21] X. Chang, F. Luo, W. Jiang, L. Zhu, J. Gao, H. He, et al., Protective activity of salidroside against ethanol-induced gastric ulcer via the MAPK/NF-kappaB pathway in vivo and in vitro, Int. Immunopharmacol. 28 (2015) 604–615. [22] L. Zhu, T. Wei, J. Gao, X. Chang, H. He, F. Luo, et al., The cardioprotective effect of salidroside against myocardial ischemia reperfusion injury in rats by inhibiting apoptosis and inflammation, Apoptosis 20 (2015) 1433–1443. [23] J. Gao, H. He, W. Jiang, X. Chang, L. Zhu, F. 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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus Zhilin Qi a,b,⁎, Shimei Qi a,b, Liefeng Ling a,b, Jun Lv a,b, Zunyong Feng b a b
Department of Biochemistry, Wannan Medical College, Wuhu, Anhui, China Anhui Province Key Laboratory of Active Biological Macro-molecules, Wuhu, Anhui, China
a r t i c l e
i n f o
Article history: Received 5 January 2016 Received in revised form 16 March 2016 Accepted 1 April 2016 Available online xxxx Keywords: Salidroside LPS JAK2-STAT3 Inflammation Acute lung injury
a b s t r a c t Salidroside (SAL) is an active ingredient isolated from the Rhodiola rosea, has potent anti-inflammatory effect, but the mechanism is still elusive. The purpose of this study is to verify the effects of SAL on LPS-induced inflammatory response and investigate the possible underlying molecular mechanism. RAW264.7 cells were preincubated with SAL for 2 h, then stimulated with or without LPS for another 16 h. The levels of TNF-α, MCP-1, IL-6, and PGE2 were detected by ELISA, and the production of NO was determined by nitrite analysis. The expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blotting. In RAW264.7 cells and murine peritoneal macrophages, the activation of signal molecules was also measured by Western blot. The nuclear translocation of STAT3 was determined by Laser confocal and nucleocytoplasmic separation experiments. Our results showed that SAL attenuated the productions of TNF-α, IL-6, MCP-1, PGE2 and NO dose dependently. SAL also suppressed LPS-induced expressions of iNOS and COX-2 significantly. Further studies revealed that SAL down-regulated the phosphorylation of JAK2-STAT3 signaling pathway and reduced the nuclear translocation of STAT3 induced by LPS in RAW264.7 cells and primary peritoneal macrophages. In addition, consistent with the results in vitro, in the model of mice acute lung injury (ALI) induced by LPS, SAL reduced the infiltration of inflammatory cells and decreased the levels of serum TNF-α and IL-6 obviously. Taken together, these data indicated that SAL exerted anti-inflammatory action via down-regulating LPS-induced activation of JAK2-STAT3 pathway and suppressing STAT3 transfer into the nucleus at least in part. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Lipopolysaccharide (LPS) also known as endotoxin, which is the main component of Gram-negative bacteria cell wall. Once released in the circulation, LPS can induce various host cells infection, especially mononuclear phagocytes that can produce pro-inflammatory cytokines including TNF-α, IL-6, and other inflammatory mediators, such as NO and PGE2, which is the production of iNOS and COX-2, respectively [1–3]. It has been reported that many inflammation related diseases are closely relevant with the accumulation of pro-inflammatory cytokines and mediators [4]. So, suppression the release of inflammatory cytokines and mediators may be an important strategy for treatment many inflammatory disorders. The JAK-STATs (Janus kinase-signal transducers and activators of transcription) is an important signaling pathway activated by many extracellular signaling ligands including LPS [2,5]. The signaling pathway has variety of biological effects such as cell proliferation, apoptosis, differentiation, and inflammation [6]. The binding of ligands and their ⁎ Corresponding author at: Department of Biochemistry, Wannan Medical College, No. 22 Wenchang West Road, Wuhu, Anhui 241002, China. E-mail address: [email protected] (Z. Qi).
http://dx.doi.org/10.1016/j.intimp.2016.04.004 1567-5769/© 2016 Elsevier B.V. All rights reserved.
receptors can induce the phosphorylations of JAKs and its downstream transcription factors STATs. Phosphorylated STATs form homo or heterodimers, transfer into the nucleus to regulate the expression of target genes associated with inflammation such as iNOS and COX-2 [7]. As we known, inflammation plays a key role in acute lung injury (ALI), and JAK-STATs signaling pathway plays an important role in the inflammation process of acute lung injury (ALI) [8]. Due to the critical role of JAKs-STATs signaling pathway in inflammatory response, inhibition the signaling pathway activation may be an interested strategy for the treatment of inflammation related diseases. Salidroside, (SAL, p-hydroxyphenethyl-β-D-glucoside), a major bioactive component extracted from Rhodiola rosea [9]. Recently, increasing studies have demonstrated that salidroside had a variety of pharmacological functions including anti-inflammatory [1,3], antitumor [10,11], neuroprotection [12,13]. The anti-inflammatory effect of salidroside on attenuation the expressions of iNOS and COX-2 and the release of inflammatory cytokines have been reported, the potent molecular mechanisms involving the inhibition of MAPKs and NF-κB signaling pathways activation [1,14]. Although salidroside has been shown to reduce inflammatory response in RAW264.7 cells upon LPS stimuli, the more detailed molecular mechanisms underlying the antiinflammatory action have not been evaluated yet. In addition, STATs
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have been implicated to be key transcription factors in both immunity and inflammatory pathways [15,16]. Thus, the aim of this study is to explore whether the anti-inflammatory activity of salidroside is mediated via suppressing JAK-STATs signal pathway activation upon LPS stimulation in RAW264.7 cells and peritoneal macrophages. In the present study, we first verified the anti-inflammatory activity of salidroside and then investigated the influence of salidroside on the activation of JAK-STATs signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Secondly, we detected the protective effects of salidroside on LPS-induced mice acute lung injury (ALI). Our results showed that salidroside could attenuate LPSinduced release of pro-inflammatory cytokines and mediators, alleviate LPS-induced ALI through suppressing of JAK2-STAT3 signal pathway and preventing the nuclear translocation of STAT3. Taken together, our study provided new sight in the anti-inflammatory mechanism of salidroside.
AG490 (20 mg/kg) 2 h prior to LPS (25 mg/kg, i.p.) treatment 12 h. After ALI, blood was collected to determine the serum levels of IL-6 and TNF-α from rats sacrificed under sodium pentobarbital (30 mg/kg, i.p.) anesthesia. In parallel experiments, after LPS stimulation for 12 h, the lungs were fixed with paraformaldehyde and then excised from rats sacrificed under sodium pentobarbital anesthesia.
2. Materials and methods
RAW264.7 cells were planted in 12-well plates and then preinclutured with different concentrations of salidroside for 2 h before 100 ng/mL LPS stimulation for another 16 h. The levels of IL-6, TNF-α, MCP-1, PGE2 in the cell culture supernatants, and the amounts of IL-6, TNF-α in the serum of rats were detected by using ELISA kit, following the manufacture's instructions. Each of the different treatments was repeated three times.
2.1. Reagents and antibodies Salidroside (purity, N 95%) was purchased from Aladdin Industrial Corporation (Shanghai, China). LPS was obtained from Sigma. 4,6diamidino-2-phenylindole (DAPI) was obtained from Invitrogen (Carlsbad, CA). The antibodies against phospho-JAK1(Y1022/1023), phospho-JAK2(Y1007/1008), JAK1, JAK2, phospho-STAT1 (Tyr701), STAT1, STAT3, COX-2, iNOS and GAPDH were all from Cell Signaling Technology (Beverly, MA, USA). The antibody against phosphoSTAT3 (Tyr705) was gained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies used for Western blotting were purchased from Rockland Immunochemical. Alexa Fluro 555 goat anti-rabbit IgG used in Laser confocal experiment was obtained from Invitrogen. 2.2. Cell culture RAW264.7 cells, purchased from Cell Bank of the Kunming (Kunming, China). were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) added with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin. 2.3. Murine peritoneal microphages extraction and culture Murine peritoneal macrophages were extracted from BALB/c mice based on the previous described method [17]. BALB/c mice (2-month-old females weighing 20 ± 2 g) were purchased from Nanjing University Experimental Animal Center and anesthetized with 2% isoflurane in oxygen via a facemask. DMEM (5–7 mL) were intraperitoneally injected, the abdomen of mice was kneaded 2– 3 min gently, then the peritoneal lavage fluid was extracted and centrifuged at 1000 rpm for 5 min at room temperature. After 2 times washing with PBS, the cells were resuspended in DMEM with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. All the cells were cultivated in six-well plates and cultured at 37 °C in 5% CO 2 . After 4 h incubation, we changed the media, removed the non-adherent cells, the cells in six-plates were used for experiment. 2.4. Animal model of acute lung injury (ALI) All procedures were approved by the Animal Care and Use Committee of Wannan Medical College. Animals were handled in accordance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. In this experiment, ALI was induced by LPS in Wistar rats (180–220 g). In briefly, rats were administered intraperitoneally with salidroside (20 mg/kg) or
2.5. Histological examination Lung tissues were rinsed thoroughly in PBS, fixed in 4% formalin, embedded in paraffin. Then the sections were stained with hematoxylin and eosin (H&E) for histological examination. Three parameters including congestion, edema and infiltration of inflammatory cells were used to evaluate the Lung injuries induced by LPS [18]. 2.6. Determination of inflammatory cytokines and medicators
2.7. Cell viability assays RAW264.7 cells were planted into 96-well plates at a density of 5000–6000 per well 24 h before treatment. Cells were treated with different dose of salidroside for 24 h, cell viability was determined by using the Cell Counting Kit-8 (Kaiji, Nanjing, China). Briefly, cells were treated with salidroside for the indicated time and then followed by incubating with 10 μL CCK-8 working solution at 37 °C and 5% CO2 for 2 h. The absorbance of each well at 450 nm was measured using MULTISKAN GO (Thermo). Each of the different treatments was done for three times, the data were presented with mean ± SD. 2.8. Nitrite analysis Cells were seeded in 96-well plates and then pre-treated with salidroside for 2 h prior to LPS stimulation or not. NO synthesis was determined spectrophotometrically by assaying the culture supernatants using the Griess reagent (Nanjing Jiancheng Bioengineering Institute). Absorbance was measured at 550 nm and the experiments were done in triplicate. 2.9. Western blot Briefly, RAW264.7 cells were rinsed with ice-cold PBS, and lysed for 30 min on ice in lysis buffer (Beyotime Biotechnology, China) supplemented with complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Lysates were centrifuged (1,2500 rpm) at 4 °C for 15 min. The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Equal amounts of protein were denatured and electrophoresed on 12% SDS-PAGE and followed by transferring onto nitrocellulose membranes (Whatman, GE Healthcare, NJ, USA). The proteins were visualized by the LI-COR Odyssey Infrared Imaging System (LI-COR) using IRDye800 flurophore conjugated secondary antibody. 2.10. Nuclear and cytoplasmic extraction RAW264.7 cells were treated with salidroside for 2 h and then stimulated with LPS or not for another 4 h. To separate the cytoplasmic and nuclear proteins, cell pellets were processed using the NE-PER nuclear
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and cytoplasmic extraction kit (Promega) according to the manufacturer's instructions.
2.11. Confocal microscopy RAW264.7 cells and primary macrophages were seeded in laser confocal small dish at 30% confluence, cells were pretreated with salidroside (200 μg/mL)for 2 h followed by LPS stimulation for another 4 h. Cells were washed with PBS, fixed with 4% polyformaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 3% bovine serum albumin in PBS for 1 h, the cells were incubated with STAT3 primary antibody overnight at 4 °C. After rinsing with PBS for three times, the cells were incubated with secondary fluorescent antibody (goat anti-rabbit IgG Alexa fluor 555 conjugates) for 1 h in the dark. After three times washing with PBS, the cells were nuclear-stained with DAPI for 3 min. Images were captured using LEICA TCS SP8 microscope.
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2.12. Statistical analysis Statistical analysis was performed with one way (ANOVA). Data were showed as means ± SD. Statistical calculations were performed by SPSS17.0. P b 0.05 was considered significant. 3. Results 3.1. Salidroside reduced the levels of pro-inflammatory cytokines and medicators induced by LPS in RAW264.7 cells As we know, the inflammatory cytokines and mediators such as TNF-α, IL-6, MCP-1, and PGE2 play an important role in inflammation. To verify the anti-inflammatory effect of salidroside on LPS induced inflammatory response, we first detected the release of IL-6, TNF-α, NO and PGE2 in LPS induced RAW264.7 cells. Fig 1A–E showed that LPS treatment obviously enhanced the levels of TNF-α, IL-6, MCP-1, PGE2
Fig. 1. Salidroside reduced the release of pro-inflammatory cytokines and mediators induced by LPS in RAW264.7 cells. (A–E) Cells were pre-incubated with salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with LPS (100 ng/mL) for another 16 h. The levels of TNF-α, IL-6, MCP-1, NO, and PGE2 were measured in the culture medium by ELISA kits or Griess reagents, respectively. (F) RAW264.7 cells were stimulated with different concentrations of salidroside for 24 h. Cell viability was evaluated using CCK-8 kit. The data were expressed as mean ± SD of three independent experiments. *P b 0.05 and **P b 0.01, versus control group.
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and NO. But, the cells pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL), the release of these cytokines and medicators was inhibited significantly in a dose dependent manner. In order to exclude the cytotoxicity of salidroside on cells, we detected the cytotoxic activity of salidroside by the CCK-8 assay. Fig 1F showed that salidroside had no cytotoxicity on cell viability even at the high dose of 400 μg/mL. 3.2. Salidroside reduced LPS-induced expressions of iNOS and COX-2 in RAW264.7 cells NO and PGE2 are two of important inflammatory mediators, which is the product of iNOS and COX-2 respectively. As above shown, salidroside attenuated the release of NO and PGE2 induced by LPS obviously. Thus, we further determined the role of salidroside on the levels of iNOS and COX-2. RAW264.7 cells were stimulated with salidroside for 2 h, then treated with LPS or not for 16 h, after the indicated time, the total proteins were extracted and subjected to Western blotting. As shown in Fig 2, LPS could induce a dramatic increase of iNOS and COX-2 proteins, while pre-treatment with salidroside significantly reduced LPS-elevated expressions of iNOS and COX-2 dose dependently. 3.3. Salidroside suppressed LPS-induced JAK2-STAT3 signal activation in RAW264.7 cells It has been reported that the STATs signaling pathway played important roles in inflammatory signaling cascades induced by LPS and other cytokines [19]. After activation, STATs form dimers, transfer into the nucleus and activate transcription of several target genes including iNOS [20]. To investigate whether the inhibitory effects of salidroside on the expressions of pro-inflammation cytokines and mediators are mediated via STATs pathway, we determined the effects of salidroside on the activation of STATs signal induced by LPS. Our previous study showed that, RAW264.7 cells stimulated with LPS, the STAT1 and STAT3 could be phosphorylated at 30 min and peaked at 4 h [2,7]. Thus, we detected the STATs phosphorylation at 4 h after LPS stimulation. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with 100 ng/mL LPS for another 4 h, Western blotting detected the phosphorylations of STAT1 and STAT3. As shown in Fig 3A, the phosphorylations of STAT1 and STAT3 were increased obviously after LPS stimulation. By contrast, the enhanced phosphorylation of STAT3 but not STAT1 was attenuated by salidroside in a dose-dependent manner. It has been reported that the STATs transcription factors were activated by their upstream kinases Janus kinases (JAKs) [6], we thus examined the effects of salidroside on the LPS-induced activation of JAKs. Our previous study showed that LPS stimulated RAW264.7 cells for 30 min, the phosphorylations of JAKs reached the maximum [2]. Then we determined the activation of JAKs after LPS stimulation for 30 min combined with or without salidroside. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, and then stimulated
Fig. 2. Salidroside suppressed the expressions of iNOS and COX-2 induced by LPS in RAW264.7 cells. Cells were pre-treated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with LPS (100 ng/mL) for 16 h. Cell lysates were collected, the levels of iNOS and COX2 were determined by Western blotting.
with LPS for 30 min, the phosphorylations of JAKs were analyzed by Western blotting. The pre-treatment with salidroside apparently attenuated LPS-induced JAK2 phosphorylation dose dependently, which was in accordance with the reduction of STAT3 phosphorylation. However, the phosphorylation of JAK1 induced by LPS was not affected by salidroside (Fig 3B). 3.4. Salidroside reduced LPS-induced nuclear translocation of STAT3 in RAW264.7 cells Since activated STAT3 should dimerize and translocate into the nucleus to regulate the transcription of target genes, we further explored whether salidroside could suppress the nuclear translocation of STAT3. Confocal microscopy experiment was used to detect the distribution of STAT3, nucleocytoplasmic separation experiment was used to confirm the level of STAT3 in the cytoplasm and nuclei. Fig 3C-D showed that STAT3 transferred into nucleus upon LPS stimulation. The cells pre-treated with salidroside, the accumulation of STAT3 in nuclei was inhibited evidently. Taken together, our results indicated that salidroside reduced LPS-induced inflammatory responses through inhibiting the activation and nuclear translocation of STAT3 in RAW264.7 cells. 3.5. Salidroside inhibited LPS-induced JAK2-STAT3 signal activation and suppressed the nuclear translocation of STAT3 in primary macrophages To further prove the role of JAK2-STAT3 signal pathway on the antiinflammatory of salidroside, we detected the effects of salidroside on LPS-induced JAK2-STAT3 activation and STAT3 nuclear translocation in murine peritoneal macrophages. Primary macrophages were preincubated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with 100 ng/mL LPS for 4 h or 30 min, The phosphorylations of JAK2 and STAT3 were detected by Western blotting. Consistent with the results of Fig 3A–B, salidroside could suppress LPS-induced JAK2-STAT3 signal pathway activation in a dose dependent manner (Fig 4A–B). In addition, we also detected the phosphorylations of JAK1 and STAT1 in primary macrophages. Our data showed that salidroside had no inhibitory effects on the phosphorylations of JAK1 and STAT1 induced by LPS (Fig 4A–B), which also consistent with the results of Fig 3A–B. Then, we detected the nuclear translocation of STAT3 in murine peritoneal macrophages. Cells were pre-treated with 200 μg/mL salidroside for 2 h, then stimulated with LPS for 4 h, the distribution of STAT3 was observed by confocal microscopy. Fig 4C showed that pre-treatment with salidroside could significantly reduce the nuclear translocation of STAT3 compared with the LPS group. 3.6. Salidroside inhibited LPS-induced acute lung injury To further certified the protective effects of salidroside on LPS induced inflammatory response, we detected the productions of serum IL-6 and TNF-α, observed the histological damage and inflammatory response in LPS induced acute lung injury model. Consistent with the results in vitro, the rats pre-treated with salidroside (20 mg/kg), the levels of serum TNF-α and IL-6 were obviously suppressed compared with the rats stimulated with LPS (Fig 5A–B). Histopathological examination showed that the histological damage, edema and inflammatory cell infiltration were alleviated obviously in the rats pre-treated with salidroside compared with the rats treated with LPS (Fig 5C). To address the relation of ALI and JAK2-STAT3 signal pathway, we used the AG490 (Sigma), JAK2 specific inhibitor as positive control. Fig 5C showed that pre-treated with AG490 (20 mg/kg), the histological damage, edema and inflammatory cell infiltration were alleviated obviously compared with the rats stimulated with LPS. Taken together, our results showed that the molecular mechanism of salidroside on LPS induced inflammatory response via suppressing JAK2-STAT3 signaling pathway in vivo and in vitro.
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Fig. 3. Salidroside inhibited LPS induced JAK2-STAT3 signal pathway activation and STAT3 nuclear translocation in RAW264.7 cells. RAW264.7 cells were pre-treated with different concentrations of salidroside (50, 100, 200 μg/mL) for 2 h, then stimulated with LPS (100 ng/mL) for the 4 h (A, C, D) or 30 min (B). The phosphorylations of STAT1, STAT3, JAK1, JAK2 and total STAT1, STAT3, JAK1, JAK2 were determined by Western blotting (A–B). (C) Nuclear and cytoplasm fractions were extracted and detected the level of STAT3 by Western blot. (D) The nuclear translocation of STAT3 was determined by confocal microscopy. Cells were fixed with 4% paraformaldehyde, permeabilized with Trixon-100, incubated with antiSTAT3 antibody, and were assessed by immunofluorescence staining with Alexa Fluro 555 goat anti-rabbit IgG antibody (red). Cell nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion As an important bioactive ingredient, Salidroside has been widely used in Chinese traditional medicine [14]. It has been reported that salidroside had a lot of pharmacological effects including anti-
inflammatory. Although recent reports have shown that salidroside played its anti-inflammatory role through inhibiting the activation of MAPKs and NF-κB signal pathways [14], more detailed mechanisms were still unclear. It has been reported that JAK-STATs signaling pathway contributed to LPS-mediated inflammatory response in lung [3].
Fig. 4. Salidroside inhibited LPS induced JAK2-STAT3 signal pathway activation and STAT3 nuclear translocation in primary macrophages. Murine peritoneal macrophages were preincubated with salidroside (50, 100, 200 μg/mL) for 2 h, then treated with 100 ng/mL LPS for 4 h (A, C) or 30 min (B), The phosphorylations of STAT1, STAT3, JAK1 and JAK2 were detected by Western blot (A–B). (C) The nuclear translocation of STAT3 was determined by confocal microscopy. Cells were fixed with paraformaldehyde, permeabilized with Trixon100, incubated with anti-STAT3 antibody, and were assessed by immunofluorescence staining with Alexa Fluro 555 goat anti-rabbit IgG (red) antibody. Cell nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Salidroside inhibited the productions of serum pro-inflammatory cytokines and attenuated the acute lung injury induced by LPS in rats. Rats were administered intraperitoneally with salidroside (20 mg/kg) or AG490 (20 mg/kg) 2 h prior to LPS (25 mg/kg, i.p.) challenge. (A–B) Blood was collected after ALI (12 h after LPS stimulation), the serum levels of IL-6 and TNF-α from rats sacrificed under sodium pentobarbital anesthesia were detected by ELISA kits. (n = 5 rats/group). Data were expressed as mean ± SD. *P b 0.05 and **P b 0.01, versus control group. (C) Lung tissues from different group rats were collected and subjected to H﹠E staining and detected by light microscopy (magnification, ×400).
So, in the present study, we investigated the possible correlation between the anti-inflammatory effect of salidroside and JAK-STATs signaling pathway. Our data showed that salidroside significantly inhibited the activation of JAK2-STAT3 signaling pathway, and suppressed the nuclear translocation of STAT3. In this study, we first verified the anti-inflammatory effect of salidroside in RAW264.7 cells, we observed that salidroside suppressed the release of TNF-α, IL-6, MCP-1, PGE2, NO and downregulated the expressions of iNOS and COX-2, which were induced by LPS. In addition, the inhibitory effect was concentration dependent (Figs. 1 and 2). Secondly, we investigated the underlying antiinflammatory molecular mechanisms of salidroside in RAW264.7 cells.
Accumulating evidences had showed that a variety of signal pathways involved in the protective effects of salidroside such as NF-κB, MAPKs and PI3K-AKT signaling [21–24]. Some studies have proved that STATs and its upstream kinases played an important role in inflammatory and immune response induced by LPS [25,26]. But, whether the signaling pathway involved in the anti-inflammatory effect of salidroside is still unclear. Our previous study showed that STAT1 and STAT3 could be phosphorylated by LPS in RAW264.7 cells [2]. After activation, STATs dimerized and translocated to the nucleus to regulate the transcription of target genes [20]. In RW264.7 cells, we found that the pre-treatment with salidroside attenuated LPS induced STAT3 phosphorylation in a dose dependent manner (Fig 3A). But, the phosphorylation of STAT1 was not affected by salidroside. Because STATs
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phosphorylation is required for their nuclear translocations and transcriptional activities [19], to demonstrate whether salidroside could suppress the nuclear translocation of STAT3, we therefore determined the distribution of STAT3 in nuclear and cytoplasm. Western blotting detected the levels of nuclear and cytoplasm proteins in RAW264.7 cells. As shown in Fig 3C, STAT3 transferred into nucleus upon LPS stimulation. However, pre-treatment with salidroside could reduce the nuclear translocation of STAT3 induced by LPS dose dependently. Confocal microscopy analysis also revealed that salidroside attenuated the STAT3 nuclear translocation clearly (Fig 3D). These results proved that salidroside could inhibit LPS-induced inflammatory response via suppressing the activation of STAT3 at least partly. We further detected the activation of JAKs, which were the upstream kinases of STAT3. Our results showed that phosphorylation of JAK2 but not JAK1 was obviously inhibited by salidroside dose dependently in LPS stimulated RAW264.7 cells (Fig 3B). Our data suggested that salidroside attenuated LPS-induced inflammatory response via suppressing the activation of JAK2-STAT3 signaling pathway. To further determine the inhibitory effect of salidroside on JAK2STAT3 signaling pathway, we detected the activation of JAK2-STAT3 signal in primary macrophages. Consistent with the results in RAW264.7 cells, Fig 4A–B showed that pretreatment with salidroside, the phosphorylations of JAK2 and STAT3 were reduced obviously. The nuclear translocation of STAT3 was also detected by confocal laser experiment in murine peritoneal macrophages. Fig 4C showed that salidroside could inhibit the nuclear translocation of STAT3. It has been reported that pre-treatment with salidroside then stimulation with LPS could attenuate inflammatory cells infiltration, interstitial edema, hemorrhage compared with LPS stimuli in lungs of mice. In addition, salidroside could significantly reduce TNF-α, IL-6, and IL-1β levels in BALF [17]. Consistent with their results, our data also showed that salidroside could reduce the inflammatory cell infiltration, attenuate the productions of serum inflammatory cytokines TNF-α and IL-6 (Fig 5A–B). Mechanistically, LPS stimulated inflammatory signaling via TLR4, resulting in potent activation of MAPKs and NF-κB. These pathways played important roles in LPS-mediated tissue damage. The results of Guan et al. have shown that salidroside inhibited the LPS-induced ALI via NF-κB signal pathway [17]. But, the study of Mariano Severgnini, et al. demonstrated that STATs and their upstream kinases, Src and JAKs were activated in lungs of mice after LPS stimulation [25]. Their research suggested that JAK-STATs signaling pathway played an important role in LPS induced ALI. In addition, it has been reported that AG490, a special inhibitor of JAK2 pretreatment ameliorated histopathologic changes and alleviated edema in trauma-induced ALI significantly [18]. These evidences suggested that LPS-induced ALI and JAK-STATs signaling pathway were mechanistically linked. To further determine the link between the JAK2-STAT3 signaling pathway and ALI, we used AG490 as positive control, consistent with their results, in Fig 5C showed that pre-treatment with AG490, the inflammatory cell infiltration and pulmonary edema were alleviated obviously compared with the LPS treatment. Similarly to AG490, salidroside was shown to significantly reduce the inflammatory response in lungs of ALI rats induced by LPS. Taken together, our results proved that salidroside had protective effects in LPS induced inflammatory response in vivo and in vitro. In summary, the present study certified that salidroside exhibited its anti-inflammatory effect via inhibiting JAK2-STAT3 signaling pathway activation and STAT3 nuclear translocation. This study provided a new perspective for the anti-inflammatory function of salidroside. Acknowledgements The work was supported by College Outstanding Young Talent Support Program Key Projects (No. gxyqZD2016173), Natural Science Research Project of Anhui Colleges and Universities (No.·KJ2016SD59), the Natural Science Research Project of Anhui Provincial Education Department (No.·KJ2013B311), National Natural Science Foundation of
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China (31301171), The Key Scientific Research Project of Wannan Medical Colleges (WK2015Z01), Wannan Medical College Doctoral Fund, Anhui Province Key Laboratory Of Active Biological Macro-Molecules (No. 1306C083008).
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